The long-term objective of our research is to understand the mechanism and regulation of atrial natriuretic factor (ANF) biosynthesis, processing, and secretion. The proposal is focused on various treatments regulate ANF release. Our approach will involve the use of several cultured cell mode] systems and combination with biochemical and molecular biological techniques. Perhaps the greatest importance will be the use of our recently described serum-free long-term cultured heart cell system that mimic ANF processing and secretion as they occur in vivo. Specific Aims: 1) To determine the cell type responsible for ANF processing, cardiocytes and cardiac mesenchymal cells (e.g. fibroblasts, endothelial cells) will cultured separately and their abilities to process endogenous (cardiocytes) or exogenous pro-ANF will be assessed. 2) The characteristics of the immunoreactive pro-thrombin associated with the heart and cultured cardiocytes will be studied using Western analysis, peptide mapping, radiosequencing, and message characterization. The possible involvement of the atrial thrombin-like material in pro-ANF processing will be assessed. 3) ANF will be expressed in several types of endocrine cells to determine whether the unusual thrombin-like co-/post-secretional cleavage at a single basic amino acid that occurs during pro-ANF maturation is a tissue- or hormone-specific phenomenon. 4) Adrenergic receptor activation, electrical pacing of contraction rate, and changes in cellular tension will be studied as models of acutely regulating ANF secretion, and it will be determined whether these treatments regulate ANF release at the level of the cardiocyte and what intracellular messengers are involved. The proposed studies will contribute new information to our knowledge of the factors that regulate plasma levels of ANF. Such information will help us understand the hemodynamic roles of ANF in both normal and pathological states, and will provide a foundation for the future rational design of ANF-related antihypertensives.